GSM7179367: U2OS DIvA exp01 ChIC 53BP1 (KIN3383.index03); Homo sapiens; OTHER
For ChIC experiments, cells were treated as described for DamID, after which nuclei were isolated and permeabilized, incubated with primary antibody, then incubated with pA-MNase (Protein A IgG-binding domain fused to micrococcal nuclease, for antibody-specific binding) and Hoechst (for DNA content staining). If the primary antibody was raised in mouse, nuclei were incubated with secondary antibody (rabbit anti-mouse) before incubation with pA-MNase. Nuclei were filtered through a 20-µm mesh before FACS. DamID on single cells was performed as previously described in detail (PMID: 32350457). Briefly, after FACS, cells were lysed and treated with proteinase K, after which methylated GATCs resulting from Dam enzyme activity were specifically digested with DpnI. Double-stranded adapters containing cell-specific barcodes and a T7 promoter were ligated to the blunt (DpnI-cleaved) DNA ends. Cells with non-overlapping barcodes were pooled together to undergo in vitro transcription (IVT), amplifying the genomic DamID-specific product in a linear manner. Library preparation was then performed on the amplified RNA, to generate molecules compatible with Illumina sequencing. ChIC was performed as previously described in detail (PMID: 36539617). After FACS, proximity-based cleaving by pA-MNase was activated (for exactly 30 min on ice), followed by inactivation and proteinase K treatment. MNase-cleaved ends were then blunted and phosphorylated, and double-stranded adapters were ligated. For one experiment, A-tailing was performed after end repair of MNase-cleaved ends, followed by ligation of T-tailed adapters. DNA molecules were then further processed for sequencing as in scDamID2. Combinatorial profiling of DamID and ChIC was performed with the same procedures of nuclei isolation, antibody treatment, FACS, and molecular preparation as for scChIC. After end repair of MNase-cleaved ends, the scDamID procedure was followed, namely DpnI digestion to enrich for Dam-methylated GATCs, adapter ligation, and subsequent library preparation steps.